| Backgrounds:Nonanastomotic biliary stricture (NABS) currently is one of the major biliarycomplications after liver transplantation, and might cause biliary cirrhosis, graft failure,re-transplantation, or death. It is the bottleneck that limits the improvement of livertransplantation efficacy. Pathologically, it is caused by the biliary fibrosis of the graft thatresults from ineffective or excessive repair of the injured bile duct epithelium. Studies showthat the independent factor that affects the biliary fibrosis after liver transplantation is thecold preservation/reperfusion injury (CPRI); however, the mechanism needs to be furtherclarified.Previous studies about the mechanisms behind the biliary fibrosis of the liver graftmainly focused on the pathway of the TGF-β1mediated hepatic stellate cell activation.Recent studies confirmed that in the presence of cytokines/proinflammatory cytokines,epithelial-mesenchymal transition (EMT) of the biliary epithelial cell (BEC) is the majorsource of the matrix synthesis cells and the critical factor leading to the liver and biliaryfibrosis. Our previous study found that, BEC EMT is an important factor in biliary fibrosisstenosis after liver transplantation, and if the key link about BEC EMT is to be intervened,biliary fibrosis may be inhibited or reversed.The equilibrium between epithelial-mesenchymal transition and mesenchymal-epithelial transition (EMT/MET) determine the repair/reverse outcomes of thetrauma/inflammatory tissues. In the latter phase of trauma and inflammation, EMT wouldcontinue to be strengthened if the proinflammatory cytokines persist, and tissue/organfibrosis occurs; in contrast, if pro-inflammatory cytokines is eliminated, MET prevails overEMT, fibrosis is reversed and wounds heal. Therefore, inhibiting EMT or promoting METfor mitigating or reversing trauma and inflammation tissue fibrosis has become a new target of anti fibrosis research. Trichorhinophalangeal syndrome type1(Trps1) is involved in theEMT/MET processes of multiple types of epithelial cells embryonic development. Therepairment processes of trauma, inflammation are the reproduction of epithelial tissureembryonic development. It has been reported that Trps1play a key role in the regulation ofrenal tubular epithelial cells, alveolar epithelial cells EMT and renal, pulmonary fibrosis.However, it’s not clear whether or not Trps1is involved in the repair process of the biliaryCPRI following the liver transplantation with the possible mechanism.Objectives:By testing the liver specimens of NABS patients, in vitro and in vivo animalexperiments, we want to prove that Trps1is involved in NABS after liver transplantation,which regulate BEC EMT negatively and thereby inhibit the process of biliary fibrosis. Thisstudy is expected to broaden the understanding about the mechanism on biliary fibrosis ofthe liver graft, and provide new strategies for clinical prevention and treatment of NABSafter liver transplantation. At the same time it could provide new clues for the theoryresearches and clinical diagnosis and treatment of the traumatic biliary stricture, intra-orextra-hepatic biliary calculus and other related diseases.Methods and Results:Part Ⅰ: Trps1, epithelial and mesenchymal markers expression in liver specimens ofNABS patientsSix liver specimens of the secondary liver transplantation due to NABS were collected,Trps1, epithelial markers (E-cadherin, CK19) and mesenchymal markers (Vimentin, α-SMA)protein expression in BEC were detected by immunohistochemistry, and collagendeposition around the bile duct was detected by Masson trichrome staining.Immunohistochemistry and Masson staining results were semi-quantitative analysis.Normal bile duct surrounding tumor tissue of hepatic cavernous hemangioma was used ascontrol. Results were assessed by the linear regression analysis:(1) the correlation of Trps1and epithelial markers (E-cadherin, CK19), mesenchymal markers (Vimentin, α-SMA)protein expression levels in bile duct of liver graft;(2) the correlation of biliary fibrosisdegree in liver graft and Trps1protein expression level.In NABS group, Trps1, epithelial markers (E-cadherin, CK19) were down-regulated orabsent, mesenchymal markers (Vimentin, α-SMA) were abnormally positive in BEC, a lot of collagen was deposited around bile duct; while in control group, Trps1, epithelialmarkers (E-cadherin, CK19) were normal expressed, mesenchymal markers (Vimentin,α-SMA) were negative in BEC, only a small amount of collagen was deposited around bileduct. Semi-quantitative immunohistochemical analysis showed differences of these variousmarkers protein expression in two groups were significant (P <0.05). Semi-quantitativeanalysis about Mssson staining results also showed differences of collagen depositionaround bile duct in two groups were significant (P <0.05). Linear regression analysisshowed that, Trps1expression was positively correlated with epithelial markers (E-cadherin,CK19)(P <0.05), whereas was negatively correlated with mesenchymal markers (Vimentin,α-SMA)(P <0.05). Biliary fibrosis of the liver graft was negatively correlated with Trps1expression (P <0.05).Part Ⅱ: In vitro experimentHuman intrahepatic biliary epithelial cells (HIBECs P5100) were dealed with in vitroimitation of CPRI, then cell morphology were observed under electron microscopy, cellmotility were compared with scratch test, mRNA expression levels of Trps1, epithelialmarkers (E-cadherin, CK19) and mesenchymal markers (Vimentin, α-SMA) were detectedby PCR and results were assessed by correlation analysis, protein expression levels ofabove markers were detected by Western blotting. Respectively, recombinant adenoviruscontaining Trps1and Trps1siRNA were transfected into HIBEC to up or down-regulatedendogenous Trps1expression, then above CPRI induction experiments were repeated,epithelial marker (E-cadherin), mesenchymal markers (Vimentin) mRNA and proteinexpression were detected by PCR and Western blotting.After HIBECs were subject to the in vitro imitation of CPRI, ovoid cells changedshape into fibroblast-like cells, most of which had spindle shape, cell migratory capabilitywas significantly enhanced (P <0.05). The mRNA and protein expression of epithelialmarkers (E-cadherin, CK19) were decreased (P <0.05), whereas mesenchymal markers(Vimentin, α-SMA) were increased (P <0.05), BEC were experienced EMT. Linearregression analysis showed that in this process, Trps1mRNA expression was positivelycorrelated with epithelial markers (E-cadherin, CK19)(P <0.05), while negativelycorrelated with mesenchymal markers (Vimentin, α-SMA)(P <0.05). Afteradenovirus-Trps1was successfully transfected into HIBEC, Trps1mRNA expression was increased. Then subject to the in vitro imitation of CPRI, the mRNA transcription andprotein expression of E-cadherin were significantly increased (P <0.05), whereas the mRNAtranscription and protein expression of Vimentin were significantly decreased (P <0.05),and CPRI mediated EMT was inhibited; in contrast, after HIBECs that were transfectedwith siRNA were subject to the in vitro imitation of CPRI, the mRNA transcription andprotein expression of E-cadherin were significantly decreased (P <0.05), whereas themRNA transcription and protein expression of Vimentin were further increased (P <0.05),and CPRI mediated EMT was strengthened.Part Ⅲ: In vivo experimentAnimal model of SD rats’ orthotopic liver transplantation with hepatic arterialreconstruction were established, the cold preservation time of donor liver were respectively1h,6h,12h, and four time points1d,3d,7d,14d after operation were select as theobservation window, the mRNA expression levels of Trps1, epithelial markers (E-cadherin,CK19) and mesenchymal markers (Vimentin, α-SMA) in bile duct were quantitativelydetected by Real-Time PCR, the protein expression levels of Trps1, epithelial marker(E-cadherin), mesenchymal marker (Vimentin) were detected by Western blotting.With the cold preservation time of donor liver extended, the mRNA and proteinexpression levels of Trps1and epithelial markers (E-cadherin, CK19) were graduallydecreased (P <0.05), the mRNA and protein expression levels of mesenchymal markers(Vimentin, α-SMA) were gradually increased (P <0.05). With the postoperative timeprolonged, the mRNA and protein expression levels of epithelial markers (E-cadherin,CK19) in the graft bile duct were gradually decreased (P <0.05), the mRNA and proteinexpression levels of mesenchymal markers (Vimentin, α-SMA) were gradually increased (P<0.05). But the mRNA and protein expression levels of Trps1were down-regulated briefly,followed by up-regulated for a longer time after the operations, which showed reducedpostoperative1d, began to rise at postoperative3d, and peaked at postoperative14d.Conclusions:1. Trps1is involved in the pathogenesis of NABS after liver transplantation, and isnegatively correlated with BEC EMT and biliary fibrosis of the liver graft.2. In vitro imitation of CPRI could mediated EMT in HIBECs; Trps1was involved inthe regulation of EMT in HIBECs, had important antagonistic effects, and might reverse the process.3. CPRI could cause Trps1expression downregulated in the postoperative SD rats’ bileduct tissues; thereby induce the EMT of BEC, which further confirm in a real diseasecondition that Trps1is a key negative regulatory factor to the EMT of BEC.4. After liver transplantation, Trps1expression levels in the SD rats’ bile duct tissueswere negatively correlated with the EMT of BEC. But the change trend of Trps1expressionis not entirely consistent with EMT process, suggesting that Trps1may inhibit biliaryfibrosis in liver graft and promote its repair. |
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